For the first time, we identify and functionally characterize spinal neurons engaged during bladder activity and define their role in coordinating autonomic and somatic components of lower urinary tract function (LUTF). LUTF requires precise coordination between autonomic pathways controlling bladder contraction and somatic pathways regulating the external urethral sphincter (EUS). Although supraspinal centers regulate aspects of LUTF, the spinal cord serves as a critical hub integrating sensory inputs and generating motor output (1). Yet, the specific spinal neuronal populations orchestrating these functions remain poorly defined.

Neurons activated during repeated bladder filling and voiding in awake animals were selectively labelled using a cFos-based transgenic mouse line (TRAP2), enabling enrichment of functionally relevant populations by flow cytometry before encapsulation and sequencing using the 10X Chromium 3’ GEM-X platform. High-resolution spatial transcriptomics using the 10X VisiumHD assay was used to further characterize and localize all transcriptionally defined populations across the lumbosacral spinal cord (n=18 L6-S1 spinal cross-sections). To define circuit connectivity of specific neuronal populations, we performed Cre-dependent viral tracing using an AAV expressing tdTomato-tagged synaptophysin, allowing visualization of presynaptic terminals. Functional relevance was assessed using tetanus toxin light chain (TeTxLC)-mediated silencing (n=6; both sexes) during 2-hour Micturition Video Thermography (MVT). For the assessment of optogenetic activation (n=4 males) of Neuromedin-S (Nms⁺) neurons in the lumbosacral cord, animals were implanted with a PE50 catheter into the bladder and stainless-steel electrodes into the EUS. Light stimulation was delivered through a 200 μm optic fiber positioned above the L6-S1 spinal cord. Under urethane anesthesia, multiple stimulation frequencies (2-20Hz) were tested while simultaneously recording bladder pressure and EUS electromyography (EMG).

Single-nucleus RNA sequencing (snRNA-seq) of the L6-S2 spinal cord generated a transcriptional atlas of ~25,000 neurons, resulting in 34 molecularly distinct populations, and we identified a previously unrecognized population of excitatory neurons characterized by the expression of Nms. Spatial transcriptomics further confirmed their possible involvement in LUTF as this population is largely restricted to the lumbosacral spinal cord and predominantly localized to the dorsal gray commissure (DGC). Anatomical tracing demonstrated that Nms⁺ neurons form synaptic contacts with parasympathetic bladder preganglionic neurons in the sacral spinal cord, sympathetic preganglionic neurons in the lower thoracic spinal cord, somatic motor neurons innervating the EUS, and supraspinal centers including Barrington’s nucleus, the parabrachial nucleus, and the periaqueductal gray. Functional perturbation revealed a critical role for these neurons in urinary control. Tetanus toxin light chain-mediated silencing (TeTxLC) resulted in complete urinary incontinence in all animals tested, characterized by loss of voluntary voiding and increased urine events (5 ± 3.03 vs 90.5 ± 45.27; Pre vs Post TeTxLC; p=0.0048; Figure 1). Conversely, optogenetic activation of Nms⁺ neurons (n=4) induced robust, frequency-dependent increases in bladder pressure (~10-15 cmH₂O) and EUS-EMG activity, with maximal responses observed at 20 Hz (Figure 2).

Nms⁺ neurons are predominantly localized to the DGC, a region implicated in lower urinary tract control (2). Their excitatory nature and synaptic contacts with EUS motor neurons support a role in promoting sphincter contraction and maintaining continence, as demonstrated by the consistent induction of urinary incontinence following neuronal silencing, and increased EUS-EMG activity upon optogenetic activation. Connectivity with supraspinal nuclei further suggests involvement in ascending sensory pathways (3). The concomitant increase in bladder pressure during stimulation indicates that Nms⁺ neurons may also contribute to coordinating voiding, supporting a broader role in integrating storage and voiding functions.

Neuromedin-S neurons define a previously unrecognized spinal circuit that coordinates bladder and sphincter activity and is essential for maintaining urinary continence, identifying a potential cellular target for therapeutic intervention in lower urinary tract dysfunction.

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Drake MJ, Fowler CJ, Griffiths D, Mayer E, Paton JF, Birder L. Neural control of the lower urinary and gastrointestinal tracts: supraspinal CNS mechanisms. Neurourol Urodyn. 2010;29(1):119-27.

Continence 19S (2026) 102496
DOI: 10.1016/j.cont.2026.102496